Two species of alpha-lactalbumin (alpha LA) are separable from rat milk, both of which are active in a lactose synthetase assay. The two alpha-LA's differ in apparent molecular weights (22,500 and 21,800 daltons respectively) and net negative charge. Enzymatically and by radioimmunoassay, alpha-LA represents about 6% of the total protein of rat milk and 0.6% of the 3-5 day lactating rat mammary gland protein. We have quantitated the mRNA for alpha-LA and casein in the lactating rat mammary gland by translating the mRNA in a wheat germ system. The in vitro synthesized alpha-LA and casein were identified on SDS-polyacrylamide gels after immuno-precipitation with rat alpha-LA or casein antibody. Thirty percent of the total protein synthesized in vitro from total cellular RNA was precipitated with alpha-LA antibody, and the remaining 70% with casein antibody. Proteins synthesized in vitro with poly A-bearing RNA isolated from total cellular RNA by either dT-cellulose or Poly(U)-sepharose columns, consisted of 40-50% alpha-LA with casein as the remainder. After fractionation of poly A RNA on sucrose gradients, alpha-LA message sediments as an 8.2 S peak and represents nearly 50% of the total active message.